A number of molecular genetic approaches are being employed to complement biochemical studies of the pathobiology of Chlamydia trachomatis. In collaboration with Harlan Caldwell and others in this laboratory, the following projects are being pursued: (1) Cloning of genes encoding outer membrane components of C. trachomatis. A variety of cloning strategies have been used in order to isolate the gene encoding surface components of C. trachomatis and C. psittaci. It is hoped that a comparison of conserved structural domains between similar surface molecules of C. trachomatis and C. psittaci will point to functionally important segments of the molecule. From the amino acid sequence of a portion of the major outer membrane protein (MOMP) of C. trachomatis, an oligonucleotide was constructed that was predicted to hybridize to the gene encoding the MOMP. Using this oligonucleotide as a DNA probe, we isolated a recombinant clone that produced a 15Kd peptide that reacted with monoclonal antibody directed against the MOMP. We have cloned larger DNA segments in order to achieve expression of the intact 40Kd MOMP but have been unsuccessful with this approach. We are currently sequencing the MOMP gene and surrounding DNA sequences. (2) DNA sequence analysis of chlamydial plasmids. The plasmids of Chlamydia are suspected of coding for gene products essential to pathogenesis. As an initial analysis of the plasmids, we have cloned the entire single plasmids from different strains and have begun DNA sequence analysis. Knowledge of the DNA sequence will allow us to manipulate the expression of open reading frame segments of the plasmid and to follow plasmid transcription.